Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts

Autor: Jacoba J. Out-Luiting, Remco van Doorn, Abdoelwaheb El Ghalbzouri, Paul P. M. van Zuijlen, Marion Rietveld, Rajiv S Raktoe, Marianna Kruithof-de Julio
Přispěvatelé: Plastic, Reconstructive and Hand Surgery, AMS - Tissue Function & Regeneration, Amsterdam Movement Sciences - Restoration and Development, Amsterdam Movement Sciences
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: Raktoe, Rajiv S; Rietveld, Marion H; Out-Luiting, Jacoba J; Kruithof-de Julio, Marianna; van Zuijlen, Paul Pm; van Doorn, Remco; Ghalbzouri, Abdoelwaheb El (2020). Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts. Scars, burns & healing, 6, p. 2059513120908857. Sage 10.1177/2059513120908857
Scars, burns & healing, 6
Raktoe, R S, Rietveld, M H, Out-Luiting, J J, Kruithof-de Julio, M, van Zuijlen, P P, van Doorn, R & Ghalbzouri, A E 2020, ' Exon skipping of TGFβRI affects signalling and ECM expression in hypertrophic scar-derived fibroblasts ', Scars, burns & healing, vol. 6, pp. 2059513120908857 . https://doi.org/10.1177/2059513120908857
Scars, Burns & Healing, Vol 6 (2020)
Scars, Burns & Healing
ISSN: 2059-5131
DOI: 10.1177/2059513120908857
Popis: Background: In burn patients, wound healing is often accompanied by hypertrophic scar (HS) development, resulting in both functional and aesthetic problems. HSs are characterised by abundant presence of myofibroblasts that contribute to overproduction of extracellular matrix (ECM) that is regulated by the TGF-β signalling pathway. Studies have shown that inhibition of TGF-β receptors in fibrotic diseases reduces the fibrotic load. In the present study, we aim to inactivate ALK5, also known as TGF-β receptor I, in human HS fibroblasts by exon skipping using antisense oligonucleotides (AONs). Methods: HS biopsies were used to isolate and set up fibroblast monocultures. AONs targeting ALK5 were supplemented to the fibroblast cultures to induce exon skipping, while pharmacological ALK5 inhibition was induced using SB431542. AON delivery in HS fibroblasts was examined using immunofluorescence (IF), while TGF-β signalling downstream targets, such as Smad2/3, PAI-1, ACTA2, COL1A1 and COL3A1, were analysed using touchdown polymerase chain reaction (PCR), quantitative PCR (qPCR), IF or western blotting. Results: Our data clearly demonstrate that AONs were successfully delivered in the nuclei of HS fibroblasts and that functional exon skipping of ALK5 took place as confirmed with touchdown PCR and qPCR. In addition, exon skipping affected the expression of ECM-related genes, such as type I/III collagens, PAI-1 and CCN2. Moreover, AON treatment did not affect the migration of HS fibroblasts in a model for wound healing. Conclusion: Exon skipping is a promising tool to modulate the TGF-β signalling pathway in HS. This would open a therapeutic window for the treatment of patients suffering from HSs.
Lay Summary In this research article, we describe the effect of exon skipping, a type of gene therapy, on fibroblasts that were isolated from hypertrophic scar biopsies. In these fibroblasts, we targeted the overactivated molecular pathway involved in the development of hypertrophic scars. Furthermore, this approach has been successfully studied in different diseases that show the same overactive molecular pathway. It is therefore of great interest to assess the effects of exon skipping in hypertrophic scars. The biopsies were retrieved from excisions performed on patients that developed post-burn hypertrophic scars. Upon arrival, the hypertrophic scar biopsies were prepared and the fibroblasts were isolated and put in culture on a Petri dish. All treatments were then added to the culture medium of the fibroblasts. In this study, we found that exon skipping was able to affect the main molecular pathway that is involved in the development of hypertrophic scars. Further, since this specific molecular pathway is of great importance in normal wound healing, we determined the effect of exon skipping on the migration of these fibroblasts. By simulating wound healing in a so-called scratch assay, we found that exon skipping does not affect fibroblast migration. Altogether, the present study shows that exon skipping has great potential as a novel treatment option in the management of hypertrophic scars. Additional research is warranted before we can start clinical trials.
Databáze: OpenAIRE
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